Confocal and multiphoton imaging techniques visualize typical structures or dynamic processes in biological samples and depend on existing autofluorescent matter in the specimen or the availability of suitable fluorescent dyes. As a consequence, new dyes are required to analyze unknown and potentially relevant details in biological samples.
Staining standard fluorescence samples is often time-consuming and expensive and may influence typical properties of living cells as they act in a chemical way. Furthermore, dyes lose intensity and alter the sample. They often cause phototoxicity, harm the specimen and consequently may influence the result of the experiment.
CARS overcomes these drawbacks by the intrinsic characteristics of the method. CARS does not require labeling because it is highly specific to molecular compounds which are based on vibrational contrast and chemical selectivity [1-5]. The crucial advantage of this method is that the sample remains almost unaffected.
With CARS, new samples that could not be stained due to unavailability of the appropriate dye are now accessible.
By integrating CARS technology into a confocal system, the drawbacks of conventional microscope techniques can be overcome. The latest commercial developments result in an easy-to-use and efficient imaging microscope for a variety of biological and non-biological samples. Two infrared laser beams which are adjusted exactly in terms of spatial and temporal properties generate brilliant CARS images at different wave numbers. The combination of a couple of CARS filters and non-descanned detectors allows the detection in the forward as well as in the backward (epi) direction. Recording of the second harmonic generation (SHG) can be done simultaneously. Combinations of conventional and high-speed scanners support the analysis of dynamic processes at video rate as well as morphological studies at high resolution.